Five Hot Topics in Refinement of Nonhuman Primate Neuroscience Research
This blog post was originally published in Laboratory Animal Science Professional magazine, a publication by the American Association for Laboratory Animal Science. Contributors include Meaghan Loy, MS, Category Director, In Vivo Services at Scientist.com, as well as Christine L. Buckmaster, BS; Denyse Levesque, DVM; Megan LaFollette, MS, PhD; Jennifer L. McMillan, BS; Mark J. Prescott, PhD; Sally Thompson-Iritani, DVM, PhD.
Nonhuman primates (NHPs) play an important role in neuroscience research and in advancing our understanding of normal neurological function, behavior, and diseases such as Parkinson’s, Alzheimer’s, and spinal cord injury. This important biomedical research is supported by continual evaluation of opportunities to improve the lives of research animals in line with the refinement principle of the 3Rs.24 The purpose of this article is to higlight and provide resources for five recent evidence-based NHP refinement advances in neuroscience. It is important to always remember that modifications need to be reviewed and implemented in partnership with the researchers, veterinary and animal care staff as well as oversight bodies (e.g., IACUC (Institutional Animal Care and Use Committee), AWERB (Animal Welfare and Ethical Review Board), NIH (National Institutes of Health) to ensure they are incorporated in a thoughtful and measured approach that delivers both improved animal welfare and optimal data.
Members of the North American 3Rs Collaborative (NA3RsC) refinement initiative have chosen to summarize developments in five hot topics of interest:
- Tips for welfare-friendly transport, chairing, and restraint
- Guidance on refining food and fluid control
- Protective caps for cranial implants
- MRI Neuroimaging
- Retirement: an optional experimental endpoint
1. Tips for Welfare-friendly Transport, Chairing, and Restraint
Traditionally, NHPs are often restrained to facilitate safe handling and obtain critical research outcomes. However, restraint may cause stress for some animals, potentially confounding study results.25,27 Therefore, it is important to refine these methods whenever possible. It is now common in many neuroscience labs to train animals to voluntarily exit their home enclosure, enter a primate chair, accept restraint, and sit in place for an extended period. In fact, in only 20 days, rhesus macaques can be trained without pole-and-collar to voluntary transfer from the home enclosure into a primate chair, and accept neck-plating.11 Overall, the chairing procedures can be improved - made faster and less stressful - through a greater understanding of operant conditioning and how to best incorporate positive reinforcement training techniques.13
Choosing an appropriate chair design can also support improved handling and animal welfare. Two main types of chairs exist: box and open. In a box chair, animals are trained to enter and extend their heads through an aperture on the top. In an open chair, animals are transferred manually or through the pole-and-collar process. In a recent survey, two-thirds of labs using restraint indicated using a ‘box
chair’ compared to one-third of labs which used an ‘open chair.’12 Literature also suggests the ‘box chair’ can improve training success, decrease training time, and reduce stress compared to the ‘open chair.’8
Alternatives to physical restraint should also be considered as recommend by the Guide for the Care and Use of Laboratory Animals.9 For example, attaching a touch screen computer tablet directly to the cage allows NHPs to work continuously and stay free-moving in their home enclosure during initial training on cognitive tasks, before moving to the laboratory for electrophysiology. The ‘Mymou’ wireless, tablet-based system incorporates real-time facial recognition, allowing the animals to remain socially housed.4 Beyond tablets, training programs can also eliminate the need for restraint. For example, researchers at the University of Minnesota were able to eliminate chair restraint after implementing a training program focusing on the handler-animal relationship including hand feeding and drinking, shifting and limb presentation.5 All of the above refinements may improve the overall well-being of animals involved in neuroscience research, bene”ting the animals, handlers, and science.
2. Guidance on Refining Food and Fluid Control
Control of food and fluid have long been used to motivate NHPs to perform certain activities for food and fluid rewards during neuroscience and behavioral studies. However, international research institutions have recognized that if food and water regulation are not conducted appropriately, they can have animal welfare implications.20 Therefore, food and fluid management require careful consideration, ethical review, and approval as well as close work with oversight and clinical staff. Fluid restriction may be particularly distressing as water is a physiological requirement. Any restriction needs to be carefully monitored for individual animals to ensure adequate food consumption and body weight/growth are maintained.1 Extensive guidance on refining these protocols is available from the NC3Rs (National Centre for the Replacement, Refinement and Reduction of Animals in Research) and APV (Association of Primate Veterinarians).1,20
For some studies, food and water restriction can be avoided or relaxed by optimizing positive reinforcement training. For example, identifying individually preferred rewards can improve performance in cognitive tasks, which can be advantageous because the NHPs may work reliably on a more relaxed fluid restriction regimen.6
In a large contract research organizations, highly preferred items such as yogurt, sports drinks, raisins, and nuts have been used to train sexually mature male macaques for procedures (such as blood sample collection). The increased time needed for training and minimal expense of treats may be outweighed by reduced animal distress and increased positive interactions between animals and technicians. Ultimately, positive reinforcement can be successful in NHPs of both sexes and all ages and elicit a variety of behaviors.19
3. Protective Caps for Cranial Implants
Neuroscience studies often require NHPs to be fitted with cranial implants to stabilize the head or gain access to the brain for scientific purpsoes (e.g., electrophysiological recordings), but wound management after surgery can be difficult. Technical and research staff from the University of Oxford and Newcastle University collaborated to develop an easily modifiable head cap to protect the sutured skin margins after surgery, restricting the monkey’s fingers from accessing the wound while allowing air to circulate to promote healing.18 Made from plastic sheeting, the cap is affixed to the implant types and head posts. Once secured, and while the animal is still anesthetized, the sheeting is molded around the implant. The protective cap, which is now being used routinely in several laboratories in the UK, EU, and USA, has reduced wound dehiscence (opening), the need for re-suturing, and the number of days antibiotics and analgesis are required. Further advances in cranial implant design, implantation and maintenance are given in the NC3Rs chronic implants wiki (https:/www.ciwiki.net).
4. MRI Neuroimaging
Magnetic resonance imaging (MRI) is playing a significant role in applying the 3Rs to neuroscience studies. For individual research facilities MRI can contribute to refinement in many ways. MRIs can be used to enhance the selection and assignment of NHPs to studies, guide the manufacture of custom-fitted recording and head fixation devices, target brain regions more accurately (e.g., for electrode insertions and compound injections), and monitor and diagnose health issues.17,21 Finally, MRI results can be combined with behavioral assessment to gauge physiological well-being to better understand the impact of neuroscience procedures on the welfare of NHPs.2
Across the entire field, NHP welfare and the 3Rs can be advanced through collaboration, open resources, resource sharing, and international harmonization. Some key resources include the International Neuroimaging Data-sharing Initiative (INDI), the PRIMatE Data Exchange (PRIME-DE) which is an open resource for NHP MRI neuroimaging data15 and the PRIMatE Resource Exchange.14 In a recently held PRIME-DE meeting entititled, Accelerating the Evolution of Nonhuman Primate Neuroimaging,23 key program priorities were decided and it was agreed additional transparency on NHP welfare standards and regulations is needed when sharing data sets. International harmonization of animal welfare standards requires mutual recognition and implementation of the 3Rs3 and this would apply to the neuroimaging community’s effort towards formulating its common set of NHP welfare regulations.
5. Retirement: An Optional Experimental Endpoint
For most NHPs, euthanasia is a common endpoint due to experimental requirements and necessary collection of post-mortem data. However, when euthanasia is not a necessary endpoint, there is an opportunity to rehome NHPs to sanctuaries.10,22 NHP rehoming is clearly of interest to the research community as evidenced by recent platform sessions during annual meetings of AALAS, the APV, and the ASP (American Society of Primatologists). Incorporating retirement and rehoming into research programs may be an opportunity to strengthen the well-being of personnel working in research animal facilities and decrease euthanasia associated stress.16,26 Some insitutions in the USA are working toward novel and sustainable options for rehoming New World and Old World NHPs when research studies are completed and are or have set up financial partnerships with specific sanctuaries or are building on-site sanctuaries.7,28
In summary, this is a high-level overview of current refinements to consider when working with NHPs to support neuroscience research. It is important to recognize that each refinement must be considered in context with the scientific objective and that personnel must be properly trained to implement each technique. Finally, all modifications ened to be reviewed and implemented in partnership with the researchers, veterinary, animal care staff, and oversight bodies (e.g. IACUC, AWERB, NIH) to ensure they are incorporated in a thoughtful and measured approach that delivers both improved animal welfare and optimal data. For additional ideas of evidence-based refinements for non-human primates visit NC3Rs or NC3RsC non-human primate research hubs.
Christine L. Buckmaster is Senior Research Staff in the Department of Comparative Medicine, Stanford University, Palo Alto, CA.
Megan LaFollette, MS, PhD, is Program Manager at the North American 3Rs Collaborative, Denver, CO.
Denyse Levesque, DVM, is a Senior Veterinarian at the Yerkes National Primate Research Center and IACUC Vice-Chair at Emory University, Atlanta, GA.
Meaghan Loy, MS, is the Category Director of In Vivo Services at Scien-tist.com, Solana Beach, CA.
Jennifer L. McMillan is Training Manager for the Division of the Animal Resources at the Yerkes National Primate Research Center, Atlanta, GA.
Mark J. Prescott, PhD, is Director of Policy and Outreach at the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), in London, UK.
Sally Thompson-Iritani, DVM, PhD, is the Assistant Vice Provost, Animal Care, Outreach &3Rs at the University of Washington in Seattle, WA.
- Association of Primate Veterinarians. [Internet]. 2014. Guidelines for use of ﬂuid regulation for nonhuman primates in biomedical re-search. [Cited 29 November 2021]. Available at: https://www.primatev-ets.org/guidance-documents
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- Butler JL, Kennerley SW. 2019. Mymou: A low-cost, wireless touch-screen system for automated training of nonhuman primates. Behav Res Methods. 51:2559 – 2572.
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- Messinger A, Sirmpilatze N, Heuer K, et al. 2021. A collaborative resource platform for non-human primate neuroimaging. NeuroImage 226. 117519.
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